PROJECT SUMMARY Stroke is the most common contributor to disability in the United States (US), with over 800,000 people affected each year. In addition to treatment with intravenous recombinant tissue plasminogen activator (rtPA), endovascular mechanical thrombectomy (MT) is now the standard of care for patients with a stroke due to proximal arterial occlusion. Unlike rtPA, however, MT is available at only a limited number of tertiary centers, with stringent time-dependent effect tied to viable ischemic penumbra. As a result, many patients in rural or congested areas need rapid transfer to a tertiary center capable of delivering MT while undergoing intravenous rtPA infusion by Helicopter Emergency Medical Services (HEMS). There is thus a significant unmet need to develop neuroprotective interventions that promote patient eligibility for MT after HEMS. A significant barrier to progress, however, is concern of whether current animal stroke models adequately translate the effect of neuroprotective interventions on an ischemic brain receiving rtPA during HEMS evacuation. That concern is justified by the uniqueness of the HEMS physiological environment, with multiple physical factors such as hypobaric changes, low frequency vibration, three-axis acceleration, and extreme noise. These physical factors may affect ischemic brain receiving rtPA in multiple and opposing ways, such as decreased oxygenation in the penumbra, enhanced or decreased clot lytic effect of rtPA, increased blood-brain-barrier (BBB) permeability and raised blood pressure. There is therefore a critical need to clarify and quantify the effect of these factors on neurological outcomes, in order to ensure that preclinical animal stroke models rigorously account for the unique physiological HEMS environment. The objective of this proposal is to measure the potential effect of HEMS physical factors on ischemic brain with respect to rtPA-reperfusion, infarct size and BBB permeability. We will use a novel experimental approach that combines a traditional stroke animal model with actual helicopter transport and vibration simulation. We have a multidisciplinary team of animal researchers and engineering, with access to a dedicated Mi2 helicopter that has been adapted and certified as a flying research platform. Mice/rats undergoing an embolic stroke will be randomized to receive the rtPA infusion simultaneously either in an actual helicopter flight, vibration simulator, or under ground-based conditions. Outcome measures will include measures of rtPA activity, cerebral blood flow, infarction size and hemorrhagic transformation, and BBB permeability at 48h and sensorimotor neurological outcome measures at 7 days, all of which will be correlated with helicopter-generated factors such as vibration, acceleration and altitude. We anticipate that this work will meaningfully transform the field of acute stroke care by understanding the overall effect of HEMS in the ischemic brain. This will lead to the establishment of adequate animal models to facilitate intervention research to improve the outcomes of patients during this critical early setting.